1. Field of the Invention
This invention relates to an antiviral pharmaceutical composition comprising a combination of a vector containing the gene encoding porcine interferon-γ (pIFN-γ) and a vector containing the gene encoding porcine interferon-α (pIFN-α), wherein the composition is capable of synergistically blocking foot and mouth disease virus (FMDV) replication in vivo, and thereby acting synergistically to protect swine, bovines, goats, and sheep against FMDV challenge at doses that do not protect against FMDV challenge when administered alone and to the method of treating swine, bovines, goats, and sheep with the antiviral composition of the invention in order to reduce the degree or rate of infection by FMDV, to reduce the severity of foot and mouth disease (FMD) or any symptom or condition resulting from infection by the FMDV in the treated animal as compared to an untreated infected animal, and preferably, to protect swine, bovines, goats, and sheep against clinical FMD.
2. Description of the Relevant Art
Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family, is the most contagious pathogen of cloven-hoofed animals including bovines, swine, sheep, and goats, and causes a rapidly-spreading, acute infection characterized by fever, lameness and vesicular lesions on the feet, tongue, snout and teats (Grubman and Baxt. 2004. Clinical Micro. Rev. 17: 465-493). In areas where FMD is enzootic, disease control is achieved by slaughter of infected animals, movement control of susceptible animals, and vaccination. The current vaccine, an inactivated whole virus antigen, is not ideally suited to eliminate FMD outbreaks from previously disease-free countries since vaccinated animals cannot be unequivocally differentiated from infected animals. As a result FMD-free countries do not import animals or animal products from countries that use this vaccine, and in the event of an outbreak in disease-free countries, the most rapid method of regaining FMD-free status and resuming international trade is to slaughter infected and susceptible animals that have been in contact with infected animals. After the 2001 FMD outbreaks in the United Kingdom and The Netherlands, it became apparent that this practice is opposed by the public. International organizations such as the Office International des Epizooties (OIE) and the world organization for animal health, as well as meat-exporting countries, now support the development and use of marker vaccines and companion diagnostic tests that will allow differentiation of vaccinated from infected animals in FMD control programs (2002. The Royal Society, London, United Kingdom; Scudamore and Harris. 2002. Rev. Sci. Tech. Off Int. Epiz. 21: 699-710). We have recently developed a novel marker FMD vaccine candidate delivered by a recombinant, replication-defective human; adenovirus type 5 vector (Ad5-FMD) that can protect both swine and cattle (Mayr et al. 1999. Virology 263: 496-506; Moraes et al. 2002. Vaccine 20: 1631-1639; Pacheco et al. 2005. Virology 337: 205-209).
More recently the above-named organizations have also come to realize that to be successful, FMD control programs should include rapid measures to limit and control disease spread. To meet these needs, they now support the development of antivirals and/or immunomodulatory molecules (2002. The Royal Society, supra).
The innate immune system provides the initial response of the host to pathogen invasion (Biron and Sen. 2001. In: Fields Virology, 4th Edition, Knipe et al. (eds), Lippincott Williams & Wilkins, Philadelphia, Pa., pages 321-351). Type I interferons (alpha/beta interferons [IFN-α/β]) are rapidly induced after virus infection and via a series of events; in paracrine and autocrine processes, they lead to the expression of hundreds of gene products some of which have antiviral activity (Der et al. 1998. Proc. Natl. Acad. Sci. USA 95: 15623-15628). However, like other viruses, FMDV has evolved multiple mechanisms to overcome the IFN-α/β response (Basler and Garcia-Sastre. 2002. Int. Rev. Immunol. 21: 305-337; Conzelmann, K.-K. 2005. J. Virol. 79: 5241-5248; de los Santos et al. 2006. J. Virol. 80: 1906-1914; Devany et al. 1988. J. Virol. 62: 4407-4409; Goodbourn et a/2000. J. Gen. Virol. 81: 2341-2364; Katze et al. 2002. Nat. Rev. Immunol. 2: 675-687; Weber et al. 2004. Viral Immunol. 17: 498-515). Nevertheless, we and others have shown that pretreatment of cells with IFN-α/β can dramatically inhibit FMDV replication (Ahl and Rump. 1976. Infect. Immun. 14: 603-606; Chinsangaram et al. 2001. J. Virol. 75: 5498-5503; Chinsangaram et al. 1999. J. Viral. 73: 9891-9898) and at least two IFN-α/β-stimulated gene products (ISGs), double-stranded RNA-dependent protein kinase (PKR) and 2′-5′ oligoadenylate synthetase (OAS)/RNase L, are involved in this process (Chinsangaram et al. 2001, supra; de los Santos et al., supra). Based on these observations, we previously constructed an Ad5 vector containing the porcine IFN-α gene (Ad5-pIFNα) as a possible method of rapidly inducing protection against FMD. Ad5-pIFNα produces high levels of biologically active IFN in infected-cell supernatants (Chinsangaram et al. 2003. J. Virol. 77: 1621-1625). Swine inoculated with Ad5-pIFNα are protected when challenged with FMDV one day later, and protection can last for 3 to 5 days (Chinsangaram et al. 2003, supra; Moraes et al. 2003. Vaccine 22: 268-279). Protection correlates with an increase in the amount of IFN-α protein in serum and the induction of PKR and OAS mRNA in white blood cells (Chinsangaram et al. 2003, supra; de Avila Botton et al. 2006. Vaccine 24: 3446-3456; Moraes et al. 2003, supra). However, since this approach has not been completely effective for cattle (Wu et al. 2003. J. Interferon Cytokine Res. 23: 371-380), we are attempting to identify new strategies to induce rapid protection.
Type II IFN (IFN-γ) is a multifunctional cytokine produced by T-helper 1 (Th1) and natural killer (NK) cells, and its biological functions include immunoregulatory, anti-neoplastic, and antiviral properties (Biron and Sen, supra). The antiviral effect of IFN-γ may be direct (intracellular) or indirect, involving effector cells of the immune system (Chesler and Reiss. 2002. Cytokine Growth Factor Rev. 13: 441-454). The antiviral activity of IFN-γ against several viruses, including herpes simplex virus, hepatitis C virus, West Nile virus, vaccinia virus, vesicular stomatitis virus (VSV), human immunodeficiency virus, and coxsackievirus, another member of the picornavirus family, has been demonstrated (Cantin et al. 1999. J. Virol. 73: 3418-3423; Frese et al. 2002. Hepatology 35: 694-703; Hartshorn et al. 1987. AIDS Res. Hum. Retroviruses 3: 125-133; Henke et al. 2001. J. Virol. 75:8187-8194; Henke et al. 2003. Virology 315: 335-344; Horwitz et al. 1999. J. Virol. 73: 1756-1766; Karupiah et al., 1990. J. Exp. Med. 172: 1495-1503; Komatsu et al. 1996. J. Neuroimmunol. 68: 101-108; Shrestha et al. 2006. J. Virol. 80: 5338-5348). Recently, indoleamine 2,3-dioxygenase (INDO) (Adams et al., 2004. J. Virol. 78: 2632-2636; Bodaghi et al. 1999. J. Immunol. 162: 957-964; Obojes et al., 2005. J. Virol. 79: 7768-7776) and inducible nitric oxide synthase (iNOS) (Saura et al. 1999. Immunity 10: 21-28; Zaragoza et al. 1997. J. Clin. Invest. 100: 1760-1767) have been identified as IFN-γ-induced gene products that have intracellular antiviral effects.
Although the signal transduction pathways elicited by each type of IFN differ, the combination of type I and type II IFNs can synergistically induce gene expression (Cheney et al. 2002. J. Virol. 76: 11148-11154; Levy et al. 1990. EMBO J. 9: 1105-1111; Matsumoto et al. 1999. Biol. Chem. 380: 699-703; Thomas and Samuel. 1992. J. Virol. 66: 2519-2522). The coactivation of the IFN signaling pathways produce an increased effect in blocking the replication of a number of viruses in vitro and/or in vivo, including coronavirus (Sainz et al. 2004. Virology 329: 11-17), herpes simplex virus (Balish et al. 1992. J. Infect. Dis. 166: 1401-1403; Sainz and Halford. 2002. J. Virol. 76: 11541-11550; Vollstedt et al. 2004. J. Virol. 78: 3846-3850), varicella-zoster virus (Desloges et al. 2005. J. Gen. Virol. 86: 1-6), cytomegalovirus (CMV; Sainz et al. 2005. Virol. J. 23: 2-14), vaccinia virus (Liu et al. 2004. FEMS Immunol. Med. Microbiol. 40: 201-206), hepatitis C virus (Okuse et al. 2005. Antiviral Res. 65: 23-34), and mouse hepatitis virus (Fuchizaki et al. 2003. J. Med. Virol. 69: 188-194).
Here, we have evaluated the antiviral effect of IFN-γ on FMDV replication and determined that a combination of IFN-α and IFN-γ can act synergistically to block FMDV replication. Constructs comprising the genes encoding pIFN-γ and pIFN-α, e.g., in separate constructs or together in one construct provide a means to deliver IFN protein, allowing animals to produce IFN-γ and IFN-α endogenously. Vectors, such as recombinant replication-defective human adenoviruses, comprising these genes are effective for delivery and expression in vivo. Here, we demonstrate the antiviral properties of IFN-γ and the synergistic effect of a combination of pIFN-α and pIFN-γ on FMDV replication in cell culture. Furthermore, our in vivo experiments indicate that swine inoculated with vectors comprising pIFN-γ and pIFN-α, at doses that alone do not protect against FMDV challenge, are completely protected against clinical disease and do not develop viremia or antibodies against viral nonstructural (NS) proteins.